Use of equol for treating androgen mediated diseases

ABSTRACT

Equol (7-hydroxy-3(4′hydroxyphenyl)-chroman), the major metabolite of the phytoestrogen daidzein, specifically binds and blocks the hormonal action of 5α-dihydrotestosterone (DHT) in vitro and in vivo. Equol can bind circulating free DHT and sequester it from the androgen receptor, thus altering growth and physiological hormone responses that are regulated by androgens. These data suggest a novel model to explain equol&#39;s biological properties. The significance of equol&#39;s ability to specifically bind and sequester DHT from the androgen receptor have important ramifications in health and disease and may indicate a broad and important usage for equol in the treatment and prevention of androgen-mediated pathologies. Thus, equol can specifically bind DHT and prevent DHT&#39;s biological actions in physiological and pathophysiological processes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. Ser. No.13/903,748, filed May 28, 2013, now issued U.S. Pat. No. 9,089,547,which is a continuation of U.S. Ser. No. 13/442,466, filed Apr. 9, 2012,now issued U.S. Pat. No. 8,450,364, which is a continuation of U.S. Ser.No. 12/572,791, filed Oct. 2, 2009, now issued U.S. Pat. No. 8,153,684,which is a continuation application of U.S. Ser. No. 10/533,045, filedOct. 20, 2005, now abandoned, which is a U.S. national stage filingunder 37 U.S.C. §371 of International Application PCT/US03/34441, filedOct. 29, 2003, which claims priority to U.S. provisional patentapplication No. 60/422,469, filed Oct. 29, 2002, contents of which areincorporated herein by reference in their entirety.

GOVERNMENT INTERESTS

This invention was made with Government support under Grant No. NS39951, awarded by the National Institute of Health (NIT, and Grant No. NRI2002-00798, awarded by the U.S. Dept. of Agriculture (USDA). TheGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

This invention relates to equol and its mechanism of action and use as atherapeutic compound for treating and preventing physiological andpathophysiological conditions mediated by androgens.

In recent years phytoestrogens have received increased investigativeattention due to their potential protective effects against age-relateddiseases (e.g. cardiovascular disease and osteoporosis) andhormone-dependent cancers (i.e., breast and prostate cancer). There arethree main classifications of phytoestrogens: 1) isoflavones (derivedprincipally from soybeans), 2) lignans (found in flaxseed in largequantities) and 3) coumestans (derived from sprouting plants likealfalfa). Of these three main classifications, human consumption ofisoflavones has the largest impact due to its availability and varietyin food products containing soy. Of the isoflavones, genistein anddaidzein are thought to exert the most potent estrogenic hormoneactivity and thus most attention has been directed toward thesemolecules (Knight D. C. et al., Obstet Gyneco, 187:897-904, (1996),Setchell, K. D. R., Am J Clin Nutr, 129:1333S-1346S (1998); Kurzer, M.S. et al., Anne Rev Nutr, 17:353-381(1997)). However, these isoflavonemolecules do not exist at high levels in their biologically active formin soy foods, but rather are at high abundance in a precursor form. Forexample, genistin, the precursor of genistein, is the glycosidic formthat contains a carbohydrate portion of the molecule. Additionally,malonylglucoside and acetylglucoside forms also are found. Theseconjugates are metabolized in the GI tract by intestinal bacteria, whichhydrolyze the carbohydrate moiety to the biologically activephytoestrogen, genistein. The same metabolic step occurs for theaglycone daidzein, which is converted from the glycosidic form daidzin.Diadzein is then further metabolized to equol in an “equol-producing”mammal. Thereafter, equol circulates in the blood stream at very highconcentrations. Equol is not normally present in the urine of mosthealthy adults unless soy is consumed. The formation of equol in vivo isexclusively dependent on intestinal microflora as evidenced from thefinding that germ-Phyto-Free animals do not excrete equol, and thatequol is not found in the plasma and urine of newborn or 4-month oldinfants fed exclusively soy foods from birth due to the fat that theintestinal flora has not yet developed in neonates. See Setchell K. D.R. et al The Lancet 1997; 350:23-27.).

The phenolic ring structures of isoflavones enable these compounds tobind estrogen receptors (ER) and mimic estrogen. Although genistein anddaidzein bind to ER, it is with a lower affinity when compared toestradiol, and with a greater affinity for ERβ than to ERα.Additionally, phytoestrogens have been reported to act like naturalselective estrogen receptor modulators (SERMs) at various tissue sitesthroughout the body. In some tissues, there is evidence thatphytoestrogens act as estrogen agonists, whereas in others, they displayantagonistic characteristics comparable to that of tamoxifen orraloxifene where SERM activity appears to be sex-hormone and genderdependent.

While the bulk of the scientific literature has focused on the naturalisoflavones in soy or clover, little has been reported on the actions oreffects of their intestinally derived metabolites.

Equol (7-hydroxy-3(4′hydroxyphenyl)-chroman) represents the majormetabolite of the phytoestrogen daidzein, one of the main isoflavonesfound abundantly in soybeans and soy-foods. Equol, however, is not aphytoestrogen, because it is not a natural constituent of plants. Equoldoes not occur naturally in any plant-based products. Rather, it is anon-steroidal isoflavone that is exclusively a product of intestinalbacterial metabolism (relatively few individuals, ˜30-40%, have themicro flora necessary to convert soy isoflavones to equol). Previousresearch with equol has identified that equol possess some weakestrogenic properties, binds sex hormone binding globulin, bindsα-fetoprotein, and has antioxidant activity. However, equol is uniqueamong the plant-derived isoflavones in that it possesses a chiral centerand as such exists as two distinct enantiomeric forms, the R- andS-enantiomers. We have shown that the S-enantiomer of equol is theexclusive equol form found in the urine and plasma of “equol-producing”mammals consuming soy, and is the only equol enantiomer made by humanintestinal bacteria. All previous studies on equol appear to have beenconducted with the racemic form of equol. There has in general been alack of appreciation that two forms of equol exist and to our knowledgeno previous study has reported on the specific actions or activity ofthe individual enantiomers. The R- and S-enantiomers conformationallydiffer, which subsequently influences their biological activity. Forexample, only the S-enantiomer of equol binds estrogen receptor (ER)with sufficient affinity to make it relevant to bind circulating equollevels reported in humans. Compared to 17β-estradiol the relativebinding affinities of the R- and S-equol enantiomer for ERα are 210.6and 49.2 fold less respectively. However, the S-equol enantiomer seemsto be largely ER□-selective with a relatively high affinity for ERβ.Enantiomer S-equol binds ERβ at approximately 20% that of 17β-estradiol[equol, Kd=0.7 nM vs. 17β-estradiol, Kd=0.15 nM], while the R-equolenantiomer binds at approximately 10(fold less. R-Equol, although notnaturally occurring, is of considerable importance because of itsability to modulate androgen-mediated processes in the body.

The prostate gland depends on androgen hormone action for itsdevelopment and growth, and the development of human benign prostatichyperplasia (BPH) clearly requires a combination of testicular androgensduring the aging process. However, testosterone is not the majorandrogen responsible for growth of the prostate. The principal prostaticandrogen is dihydrotestosterone (DHT), as evidenced by currenttreatments of prostatic cancer are directed toward reducing DHT with5α-reductase inhibitors. Although not elevated in human BPH, DHT levelsin the prostate remain at a normal level with aging, despite a decreasein the plasma testosterone. Testosterone is converted to DHT by5α-reductase in prostatic stromal and basal cells. DHT is primarilyresponsible for prostate development and the pathogenesis of BPH.Inhibitors of 5α-reductase reduce prostate size by 20% to 30%. Thisreduction in glandular tissue is achieved by the induction of apoptosis,which is histologically manifested by ductal atrophy. 5α-reductaseoccurs as 2 isoforms, type 1 and type 2, with the prostate expressingpredominantly the type-2 isoform, and the liver and skin expressingprimarily the type-I isoform. Patients have been identified withdeficiencies in the type-2 5α-reductase, but not type 1. Knockout micewith the type-2 5α-reductase null-mutation demonstrate a phenotypesimilar to that seen in men with 5α-reductase deficiency. Type-15α-reductase knockout male mice are phenotypically normal with respectto reproductive function. Enzymatic activity for 5α-reductase orimmunohistochemical detection has been noted in other genitourinarytissues, such as the epididymis, testes, gubernaculum, and corporalcavernosal tissue.

Quantitatively, women secrete greater amounts of androgen than ofestrogen. The major circulating steroids generally classified asandrogens include dehydroepiandrosterone sulphate (DHEAS),dehydroepiandrosterone (DHEA), androstenedione (A), testosterone (T),and DHT in descending order of serum concentration, though only thelatter two bind the androgen receptor to a significant degree. The otherthree steroids are better considered as pro-androgens. −DHT is primarilya peripheral product of testosterone metabolism. Testosterone circulatesboth in its free form, and bound to protein including albumin and sexsteroid hormone-binding globulin (SHBG), the levels of which are animportant determinant of free testosterone concentration. Thepostmenopausal ovary is an androgen-secreting organ and the levels oftestosterone are not directly influenced by the menopausal transition orthe occurrence of menopause.

The work of some research has focused on the development of steroidalcompounds for the treatment of androgen dependent diseases such as:hirsutism, androgenic alopecia, benign prostatic hyperplasia (BPH) andprostate cancer. DHT has been implicated as a causative factor in theprogression of these diseases, largely through the clinical evaluationof males who are genetically deficient of steroid 5α-reductase enzyme.As a result of such studies, the inhibition of this enzyme has become apharmacological strategy for the design and synthesis of newantiandrogenic drugs. However, it is unclear whether inhibition of5α-reductase will have a deleterious impact on the system, as evidencedby contraindications arising from reported side effects of conventionaltreatments using 5α-reeducates inhibitors. The development of differentstrategies that target the inhibition of DHT effects would be a majoradvance in the therapy of androgen-mediated conditions.

Despite the recent gains in understanding the pharmacology of equol asit pertains to estrogen actions, our research showing potentantiandrogen effects of equol is unique and novel and opens newapproaches to preventing or treating androgen-related conditions.Binding or sequestering DHT would provide a means for inhibiting itseffect on DHT-sensitive tissues. There is no known ligand that isspecific for DHT, but such an agent would have distinct advantages overnon-discriminatory compounds that target the androgen receptor directlyor the enzymes involved in androgen synthesis.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a method of modulating physiologicaland pathophysiological conditions mediated by androgens in a mammal,comprising the step of administering to the mammal an effective amountof an enantiomeric equol that can bind with free 5α-dihydrotestosterone,thereby inhibiting the binding of 5α-dihydrotestosterone with theandrogen receptors (AR) in the mammal and mediating the conditionsmediated by the androgen.

The present invention also relates to a method of treating andpreventing an androgen-related disease in a mammal, comprising the stepof administering to the mammal an effective amount of an enantiomericequol that can bind with free 5α-dihydrotestosterone, thereby inhibitingthe binding of the 5α-dihydrotestosterone with the androgen receptors inthe mammal.

The present invention further relates to a method of modulating androgenhormone activity in a mammal, comprising the step of administering tothe mammal an effective amount of an enantiomeric equol that can bindwith free 5α-dihydrotestosterone, thereby modulating the binding of5α-dihydrotestosterone with the androgen receptors in the mammal.

The present invention additionally relates to a method of preventing DHTbinding to the AR by contacting the DHT with an enantiomeric equol priorto the binding of DHT and AR.

The present invention relates to a method of treating and preventing acombination of an androgen-related condition and an estrogen-relatedcondition in a mammal, comprising the step of administering to a mammalan effective amount of a mixture of R-equol and S-equol, that can bindwith free 5α-dihydrotestosterone, and with free 5α-dihydrotestosteroneand the estrogen receptor, respectively, thereby inhibiting the bindingof the 5α-dihydrotestosterone with the androgen receptors, and affectingbinding of the estrogen receptors.

The present invention also relates to a method of modulating age-relatedandrogen/estrogen hormonal balances, comprising the steps of: 1)determining a mammal's endocrine androgen/estrogen hormone balance, 2)administering to the mammal an effective amount of a mixture of R-equoland S-equol, that can modulate the hormone balance of5α-dihydrotestosterone and estrogen.

The present invention further relates to a use of an enantiomeric equolto bind in vivo free DHT, for modulating physiological andpathophysiological conditions mediated by androgens in a mammal.

The present invention also relates to a method of regulating the levelof LH in vivo in a mammal by contacting the DHT of the mammal withenantiomeric equol.

The present invention relates to a use of enantiomeric equol as adiagnostic agent for physiological and pathophysiological conditionsmediated by androgens/androgen-related disorders affected by anestrogenic/androgenic imbalance.

The present invention further relates to a use of equol in a competitivebinding assay, the assay comprising the steps of: 1) providing anandrogen receptor, 2) providing a complex of DHT-enantiomeric equol, 3)providing a test substance comprising an androgen binding moiety, 4)contacting and competing for the DHT-enantiomeric equol complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures of S-equol and R-equol enantiomers.

FIG. 2 shows an appearance/disappearance plot of R-equol in plasma afteroral administration of R-equol to a healthy adult.

FIG. 3 shows a mass chromatogram of the elution of the equol enantiomersfrom a sample of urine from an adult consuming soy food, comparedagainst pure enantiomeric standards that had been characterized byoptical dichroism.

FIG. 4 shows the GC-MS analysis of the trimethylsilyl ether derivativeof synthesized product.

FIG. 5 shows a mass chromatogram of a chiral separation of S-equol andR-equol from a racemic mixture.

FIG. 6A shows prostate weight for in intact male rats subcutaneouslyinjected with DMSO or equol.

FIG. 6B shows lutenizing hormone (LH) for in intact male ratssubcutaneously injected with DMSO or equol

FIG. 7 shows a distinct peak in [3H] DHT+equol but not [3H] DHT alone.

FIG. 8A shows two distinct peaks in [3H]-DHT+equol incubated withprostate (A), while,

FIG. 8B shows only a single peak is present in [3H]-DHT incubated withprostate (B).

FIG. 9 shows the specific binding of equol to [3H]-DHT.

FIG. 10A shows prostate weight in gonadectomized (GDX) male rats scinjected with DMSO, DHT, equol, or both DHT and equol.

FIG. 10B shows plasma LH in gonadectomized (GDX) male rats sc injectedwith DMSO, DHT, equol, or both DHT and equol.

FIG. 11 shows plasma DHT levels in rats treated with DMSO, DHTP, equol,or both DHTP and equol.

FIG. 12 shows the histological effects of equol in the prostate gland ofGDX (A-D) and intact (E & F) rats treated with either Trent: DMSO (A &E), equol (B & F), DHT (C), or DHT plus equol (D).

FIG. 13 shows the histological effects of equol on the epididymis ofintact rats treated with DMSO (A) or equol (B).

FIG. 14 shows body weight in male rats fed either an isoflavone-rich(Phyto-600) or a phytoestrogen-free (Phyto-Free) diet.

FIG. 15 shows the white adipose tissue mass in male rats fed either aPhyto-600 or Phyto-Free diet.

FIGS. 16A and 16B show food and water intake in male rats fed aPhyto-600 or a Phyto-Free diet, respectively.

FIGS. 17A and 17B show plasma leptin and insulin levels from male ratsfed a Phyto-600 or a Phyto-Free diet, respectively.

FIG. 18 shows serum glucose levels from male rats (non-fasting) fedeither a Phyto-600 or Phyto-Free diet.

FIG. 19 shows thyroid (T3) serum levels in male rats fed either aPhyto-600 or Phyto-Free diet.

FIG. 20 shows body weights of female rats fed either a Phyto-600 orPhyto-Free diet.

FIG. 21 shows the white adipose tissue mass from female rats fed eithera Phyto-600 or a Phyto-Free diet.

FIG. 22 shows serum glucose levels from female rats fed either aPhyto-600 or Phyto-Free diet.

FIG. 23 shows serum T3 levels from female rats fed either a Phyto-600 orPhyto-Free diet.

FIG. 24 shows the body weights of female rats fed either a Phyto-60) ora Phyto-Free diet after 50 days of age.

FIG. 25 shows the white adipose tissue mass of female rats fed either aPhyto-600 or a Phyto-Free diet after 50 days of age.

FIG. 26 shows the serum leptin levels of female rats fed either aPhyto-600 or a Phyto-Free diet after 50 days of age.

FIG. 27 shows the serum insulin levels of female rats fed either aPhyto-600 or a Phyto-Free diet after 50 days of age.

FIG. 28 shows body weights of OVX rats fed either a Phyto-600 (blackbars) or a Phyto-Free (white bars) diet after and placed on a behavioralestrus induction regimen.

FIG. 29 shows the white adipose tissue mass of OVX rats fed either aPhyto-600 or a Phyto-Free diet.

FIG. 30 shows the serum leptin levels of OVX rats fed either a Phyto-600or a Phyto-Free diet.

FIG. 31 shows the body weights of 112-day-old male rats fed AIN-76,Phyto-Free, Phyto-200, or Phyto-600 diets.

FIG. 32 shows the body weights of 279-day-old male rats fed AIN-76,Phyto-Free, Phyto-200, or Phyto-600 diets.

FIG. 33 shows the body weights of 350-day-old male rats fed AIN-76,Phyto-Free, Phyto-200, or Phyto-600 diets.

FIG. 34 shows the adipose tissue mass from 350-day-old male rats fedAIN-76, Phyto-Free, Phyto-200, or Phyto-60) diets.

FIG. 35 shows serum insulin levels in 350-day-old male rats fed AIN-76,Phyto-Free, Phyto-200, or Phyto-600 diets.

FIG. 36 shows serum leptin levels in 350-day-old male rats fed AIN-76,Phyto-Free, Phyto-200, or Phyto-600 diets.

FIG. 37 shows body weights of 112-day-old female rats fed AIN-76,Phyto-Free, Phyto-200, or Phyto-600 diets.

FIG. 38 shows body weights of 279-day-old female rats fed AIN-76,Phyto-Free, Phyto-200, or Phyto-600 diets.

FIG. 39 shows body weights of 145-day-old male Noble rats fed Phyto-Freeor Phyto-600 diets.

FIG. 40 shows white adipose tissue mass from 145-day-old male Noble ratsfed Phyto-Free or Phyto-600 diets.

FIG. 41 shows body weights of 145-day-old female Noble rats fedPhyto-Free or Phyto-600 diets.

FIG. 42 shows white adipose tissue mass from 145-day-old female Noblerats fed Phyto-Free or Phyto-600 diets.

FIG. 43 shows baseline body weights of three groups of rats on aPhyto-Free diet prior to receiving equol injections.

FIG. 44 shows body weights of three groups of rats after 21 days on aPhyto-Free diet prior to receiving equol or vehicle injections.

FIG. 45 shows body weights of three groups of rats on a Phyto-Free diet7 days after receiving equol or vehicle injections.

FIG. 46 shows body weights of three groups of rats on a Phyto-Free diet15 days after receiving equol or vehicle injections.

FIG. 47 shows body weights of three groups of rats on a Phyto-Free diet22 days after receiving equol or vehicle injections.

FIG. 48 shows body weights of three groups of rats on a Phyto-Free diet28 days after receiving equol or vehicle injections.

FIG. 49 shows adipose tissue mass from three groups of rats on aPhyto-Free diet 28 days after receiving equol or vehicle injections.

FIG. 50 shows testes weight from three groups of rats on a Phyto-Freediet 28 days after receiving equol or vehicle injections.

FIG. 51 shows number elevated-plus maze anxiety-related behavior(entries into open arms) of 300-day-old male rats fed 4 different diets.

FIG. 52 shows elevated-plus maze anxiety-related behavior (time in openarms) of 300-day-old male rats fed 4 different diets.

FIG. 53 shows elevated-plus maze anxiety-related behavior (entries intoopen arms) of 330-day-old female rats fed 4 different diets.

FIG. 54 shows elevated-plus maze anxiety-related behavior (time in openarms) of 330-day-old female rats fed 4 different diets.

FIG. 55 shows serum isoflavone levels in 300-day-old male rats fed 4different diets.

FIG. 56 shows serum isoflavone levels in 330-day-old female rats fed 4different diets.

FIG. 57 shows the observed change in BMI for individuals after 5 weeksof strict adherence to a diet containing isoflavones.

DETAILED DESCRIPTION OF THE INVENTION

A novel mechanism of action for equol has been identified with importantramifications in health and disease and which indicates a broad andimportant usage for equol in the treatment of androgen mediatedpathologies. Equol can act as an anti-androgen. The anti-androgenicproperties of equol are unique in that equol does not bind the androgenreceptor (AR) but specifically binds 5α-dihydrotestosterone (DHT) withhigh affinity and thereby prevents DHT from binding the AR. Furthermore,both the R- and S-enantiomers of equol specifically bind DHT, sequesterDHT from the AR and block DHT's actions in physiological processes invivo. Racemic equol, which constitutes R-equol and S-equol and R-equolor S-equol alone, selectively bind DHT.

In mammals, there are two principal androgens, testosterone and its5α-reduced metabolite, DHT. DHT is recognized as the most potentandrogen in the mammalian body. The AR, which is encoded by asingle-copy gene located on the human X-chromosome, specificallymediates the actions of androgens. Although both testosterone and DHTbind the AR, certain tissues (i.e. prostate gland, hair follicles, etc.)that are only slightly influenced by testosterone are greatly influencedby DHT. Furthermore, DHT has been implicated in a number of diseases anddisorders. Because equol specifically binds and prevents the actions ofDHT, there is an indication for a broad and important usage for equol inthe treatment of androgen-mediated pathologies.

Equol has a structure similar to the steroidal estrogen estradiol. Equolis unique among the isoflavones in that it possesses a chiral center andas such exists as two distinct enantiomeric forms, the R- andS-enantiomers. All previous studies on equol appear to have beenconducted with the racemic form of equol. There has in general been alack of appreciation that two forms of equol exist and to our knowledgeno previous study has reported on the specific actions or activity ofthe individual enantiomers. R- and S-equol specifically bind5α-dihydrotestone (DHT). Equol racemic, R-equol or S-equol does not bindthe androgen receptor. R-equol does not bind the estrogen receptorsystem. S-equol only binds ERβ (with an affinity approximately 5-timesless than 17β-estradiol). Thus, R- and S-equol have SERM-like propertiesalong with having the capability to selectively bind the most potentcirculating androgen, DHT.

Most of the interest in soy and its constituent isoflavone, genistein,and to a lesser extent daidzein, has focused on either their estrogenicactions, or other non-hormonal actions such as their affects on enzymes,growth factors or cytokines, or their antioxidant actions. Neverpreviously has there been any discussion of the potential antiandrogenactions for isoflavones and rarely is the enantiomeric forms of equoleven mentioned. The present invention addresses the effects of theenantiomeric forms of equol and specifically, the ability of bothS-equol, the natural metabolite of daidzein, and R-equol to antagonizethe actions of the potent androgen dihydrotestosterone, DHT. Sucheffects open up novel possibilities for dietary, nutraceutical, andpharmacological approaches to prevention and treatment of disease wherethe potent androgen DHT plays a detrimental role. This includes but isnot restricted to prostate cancer, skin diseases, hair loss, andobesity. Additionally, the estrogenic actions of S-equol can also be ofbenefit in treating or preventing prostate cancer because the combinedactions of equol acting at the estrogen receptor level and as anantiandrogen.

Equol Binds with DHT

Equol (7-hydroxy-3(4′hydroxyphenyl)-chroman) represents the majormetabolite of phytoestrogens daidzin and daidzein, isoflavones foundabundantly in soybeans and soy-foods, and is an important biologicallyactive molecule. In animals fed a phytoestrogen-rich diet, the majorcirculating isoflavone is equol, which accounts for 70-90% of the totalcirculating isoflavone levels. The present invention discloses a novelmodel of equol's biological properties. In binding studies, equolenantiomers specifically bind 5α-dihydrotestosterone (DHT), but nottestosterone, DHEA or estrogen. By doing so, equol sequesters DHT fromthe androgen receptor without directly binding the androgen receptoritself. In vivo studies demonstrate that equol treatment of intact malerats significantly decreased prostate and epididymis but not testesweights. In castrated male rats treated with DHT after administeringequol, equol blocked DHT's trophic effects on the prostate gland and itsnegative feedback effects on plasma luteinizing hormone (LH) levels.

It has been found that equol can act as an anti-androgen, byspecifically binding DHT and preventing DHT from binding to the androgenreceptor (AR) without itself binding the AR. It also has been shown thatDHT that has already been bound to the AR will not be competitivelybound by enantiomeric equol. Therefore, one embodiment of the presentinvention is a method of preventing DHT binding to the AR by contactingDHT with equol prior to DHT-AR binding occurs. The enantiomeric equolmay be brought into contact with the DHT in vitro or in vivo. When theDHT is to be contacted in vivo, the equol may be administered by anyroute that allows absorption of equol to the blood stream. Biologicallyavailable DHT is free and unbound by any native ligand prior to bindingwith equol.

Reproductive organs such as the prostate and epididymis are known to beunder androgenic control. Previous data has shown that before puberty,when circulating androgen levels are very low, rats fed a dietcontaining high levels of soy-derived isoflavones have prostate weightsthat are not altered by the consumption of this diet. However, afterpuberty when androgen levels increase, prostate weights aresignificantly decreased in phytoestrogen-rich-diet fed rats compared toanimals fed a phytoestrogen-free diet. These data are similar to thepresent findings that equol-treated intact rats display significantdecreases in prostate and epididymis weights (without alterations intestes or pituitary weights during short-term studies). Notably, if theprostate and epididymal values are standardized to body weight (per 100grams) the ratios are still significantly different betweenequol-treated and control values. Equol also blocked DHT's androgenictrophic influence on the prostate and epididymis, without significantlyaltering testosterone levels.

DHT has negative feedback effects on circulating plasma levels ofluteinizing hormone (LH). Equol significantly increases LH levels bybinding DHT and preventing this feedback effect. Equol completelyreverses the inhibitory action of DHT on LH levels in gonadectomized(GDX) males, whereas DHT plus equol-treated male rats display LH levelssimilar to that of control values. These data further suggest that equolhas the specific ability to bind DHT, presumably in the bloodcirculation system, and block the hormonal action of DHT in suppressingLH production or secretion. Therefore an embodiment of the presentinvention is a method of modulating LH levels in an individual bycontacting the DHT of the individual with enantiomeric equol. The equolcan be administered by any route that allows absorption of equol to theblood stream, with the amount administered in accordance with the natureof the ailment to be treated and size of the individual.

The Structure of Equol

Equol is distinct from most isoflavones in having a chiral center due tothe lack of a double bond in the heterocyclic ring. The phytoestrogenisoflavones from soy (daidzein, glycitein and genistein), clover(formononetin and biochanin A), and kudzu, (peurarin), do not have achiral center. FIG. 1 shows the chemical structures of R-equol andS-equol.

The R- and S-enantiomers conformationally differ and this is predictedto influence how an equol enantiomer fits into the binding site in thecavity of the dimerized ER complex, and how it binds with DHT.

Approximately 50% of equol circulates in the free or unbound form inhumans, and this is considerably greater than the proportion of freedaidzein (18.7%) or estradiol (4.6%) in plasma. Since it is the unboundfraction that is available for receptor occupancy, and presumably forbinding DHT, this would effectively contribute to enhancing the overallpotency of equol.

Compositions Containing Equol

The present invention includes a composition having an at leastphysiological acceptable quantity of equol that is able to bind andsequester free DHT (but not testosterone or DHEA) thereby preventing itbinding to the androgen receptor following administration to anindividual thereby having important ramifications in health and diseaseand a broad and important use in the treatment of androgen-mediatedpathologies.

A composition containing S-equol, R-equol, a racemic equol mixture, or anon-racemic equol mixture, can be made for oral consumption. Thecomposition or a product containing the composition can be a marketed orinstitutional food product, a pharmaceutical, and an OTC medicament. Afood composition can comprise at least 1 mg, and typically up to 200 mg,enantiomeric equol or equol mixtures, per serving. Anorally-administered medicament can comprise at least 1 mg, and typicallyup to 200 mg, enantiomeric equol or equol mixture, per dose. A productfor topical application can comprise at least 0.1%, and up to 10%, byweight S-equol, or R-equol, or enantiomeric mixtures. A topicalcomposition of the present invention can include other cosmetic andpharmaceutical actives and excipients. Such suitable cosmetic andpharmaceutical agents include, but are not limited to, antifungals,vitamins, anti-inflammatory agents, antimicrobials, analgesics, nitricoxide synthase inhibitors, insect repellents, self-tanning agents,surfactants, moisturizers, stabilizers, preservatives, antiseptics,thickeners, lubricants, humectants, chelating agents, skin penetrationenhancers, emollients, fragrances and colorants.

An enantiomeric equol can also be an enantiomeric equol conjugate,conjugated at the C-4′ or the C-7 position with a conjugate selectedfrom the group consisting of glucuronide, sulfate, acetate, propionate,glucoside, acetyl-glucoside, malonyl-glucoside, and mixtures thereof.

A composition or preparation comprising enantiomeric or mixture ofequol, for administering to subjects for the treatment and/or preventionof, or for reducing the predisposition to, androgen-related diseases andconditions related thereto, can also comprise one or morepharmaceutically acceptable adjuvants, carriers and/or excipients.Pharmaceutically acceptable adjuvants, carriers and/or excipients arewell known in the art, for example as described in the Handbook ofPharmaceutical Excipients, second edition, American PharmaceuticalAssociation, 1994 (incorporated herein by reference). The compositioncan be administered in the form of tablets, capsules, powders forreconstitution, syrups, food (such as food bars, biscuits, snack foodsand other standard food forms well known in the art), or in drinkformulations. Drinks can contain flavoring, buffers and the like.

The composition of the invention can comprise a non-racemic mixture ofS-equol and R-equol, having an EE for S-equol of more than 0% and lessthan 90%. A composition that has an EE of 0% is a 50:50 racemic mixtureof the two enantiomers. The composition can be made directly from aracemic mixture, by an incomplete separation and removal of either theR-equol or S-equol enantiomer from the racemic mixture. The compositioncan also be made by combining a first equol component comprising amixture (either a non-racemic or racemic mixture) of equol, with asecond component comprising a composition consisting essentially ofS-equol or R-equol. This produces a non-racemic composition that has anexcess of S-equol or R-equol. Depending upon the specific benefit orindication for the R-equol component and the S-equol component in acomposition, a composition can be prepared comprising S-equol andR-equol at a ratio of S-equol to R-equol from greater than about 50:50to about 99.5:1, more typically about 51:49 to about 99:1, and from lessthan about 50:50 to about 1:99.5, more typically about 49:51 to about1:99.

Compositions suitable for oral administration can be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the extract; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion.

The composition typically does not comprise a significant amount of anyother androgen-receptor binding compound.

Identifying Equol Producers and Non-Equol Producers

Equol is formed following the hydrolysis of the glycoside conjugates ofdaidzein from soy, and the methoxylated isoflavone formononetin, or itsglycosidic conjugates found in clover. Once formed, equol appears to bemetabolically inert, undergoing no further biotransformation, save phaseII metabolism or a minor degree of additional hydroxylation in theliver. As with daidzein and genistein, the predominant phase IIreactions are glucuronidation and, to a minor extent, sulfation.Following the original discovery that equol's presence in urine was afunction of soy food ingestion, it was observed that approximately50-70% of the adult population did not excrete equol in urine even whenchallenged daily with soy foods, for reasons that are unclear.Furthermore, even when the pure isoflavone compounds are administered,thereby removing any influence of the food matrix, it has been shownthat many people do not convert daidzein to equol. This phenomenon hasled to the terminology of a person being an ‘equol-producer’ or‘non-equol producer’ (or ‘poor equol-producer’) to describe these twodistinct populations.

Cut-off values have been empirically derived permitting assignment ofindividuals to either of these categories. People who have plasma equolconcentrations of less than 10 ng/mL (40 nmol/L) can be classified as‘non-equol producers’ and where levels are above 10 ng/mL (40 nmol/L)this defines ‘equol producers’. This distinction can also be derivedfrom the levels in urine, an equol producer being someone excretinggreater than 1000 nmol/L. Although the excretion of equol is highlyvariable among individuals there is a large demarcation between thosethat can produce equol and those that cannot, consistent with aprecursor-product relationship in enzyme kinetics catalyzing thereaction. There is consequently an inverse relationship between urinarydaidzein and equol levels, and thus far no significant genderdifferences have been defined.

Preparation and Isolation of Equol Enantiomers

Enantiomeric equol can be prepared per se or as the racemic mixture.Chemical synthesis routes can be used to produce the racemic mixture ingood yields. In a typical synthesis process, standard chemicaltreatments are used to hydrogenate the double-bond of the heterocyclicring and to remove the carbonyl at position C-3. Typical startingmaterials are isoflavones such as daidzein, genistein, glycitein,peurarin, formononetin and biochanin A and their glucoside conjugates.Any conjugated form would be reduced to its aglycon by hydrolysis.Suitable solvents for the reaction include organic acids such as glacialacetic acid, lower alcohols such as isopropanol, and mixtures thereof.Reduction catalysts typically employed include Palladium, such as 10% Pdon charcoal. Reactions can run at temperatures from ambient to 60° C.,with pressures ranging from slightly above ambient, up to 200 psig (14atm. gauge), and with reaction times of up to 30 hours or more.

After reaction completion, the catalyst is removed and any filtrateevaporated. The crude residue is purified, typically by chromatographyemploying a silica gel column, with an eluent comprising C2-C4 alcohols,C3-C7 alkanes, and mixtures thereof. The purified residue can becrystallized from n-hexane to produce (±)equol as a pure product,typically of at least 99%, with a yield typically of at least 75%. Theequol crystallized product is colorless, not hygroscopic, and stable inair, and does not decompose during the final filtration procedure.

Method for the Isolation of the Individual R- and S-Enantiomers fromRacemic Equol

A racemic mixture of equol can be separated into its two distinctenantiomers using a chiral-phase column with a mobile phase comprising aC4-C8 alkyl and a C2-C4 alcohol. A typical example of a chiral-phasecolumn is a Chiralcel OD column or OJ column, supplied by DaicelChemical Industries Ltd. A preferred example of a mobile phase comprises70% hexane and 30% ethanol. After a first period of time from passingthe racemic mixture into the inlet, the time period depending upon thetype of column, type of eluent, eluent flow rate, temperature, and massof the racemic mixture, a first effluent is collected from an outlet ofthe HPLC column. The first eluent is typically S-equol. After a secondperiod of time from passing the racemic mixture into the inlet, thesecond eluent R-equol is obtained. The elution of an equol enantiomerfrom the column can be detected by UV absorbance at 260-280 nm or by amore specific detection system such as a mass spectrometer andmonitoring of ions specific to equol.

Biological Production of S-Equol

S-equol can be produced biologically in bulk using conventional foodtechnology. A base solution media, food product or plant extract can beprovided that comprises daidzein or another related isoflavone fromwhich daidzein can be derived. The daidzein or other isoflavone can beconverted to S-equol by a standard bacterial or enzyme fermentationprocess, to provide a bulk solution, food product or plant extract thatcomprises S-equol.

Conversion of daidzein to equol involves three major steps: 1)hydrolysis of any glucoside conjugate group, 2) conversion of theisoflavone aglycons to a dihydro-intermediate, and 3) conversion of thedihydro-intermediate to equol. The metabolic pathway and enzymes foreach of the three steps required may not necessarily be present in onebacterium. Anecdotal evidence from human studies suggests that there maybe one or more bacteria that act in conjunction to perform thesereactions, as evidenced from the fact that often dihydrodaidzein can bepresent in significant amounts in plasma and urine yet equol may be lowor barely detectable. Although equol may be produced from daidzein by asingle organism it is believed that better or more efficient conversioncan be achieved when using a mixture of bacterial species, each with itsown metabolic profile. Important conditions for effective conversion toS-equol include the selection of the bacterial organism or mixture oforganisms, the temperature of incubation, and the amount of oxygenavailable to the organisms. These conditions can be optimized bytechniques well known to persons skilled in this art. The organisms usedto effect this change can be inactivated by standard techniques used inthe food industry or, alternately, allowed to remain in an active statein the product.

Typically, one or more bacterial strains are required to convert thedaidzein (or other related isoflavone) through intermediate products toS-equol, which generally involves one or more of the three majorreactions: the conversion of isoflavone glycone to aglycon isoflavone:the conversion of aglycon isoflavone to dihydro isoflavone; and theconversion of dihydro isoflavone to the product, equol. For example, amixed culture of organisms isolated from equine feces or a mixed cultureof organisms derived from the gastrointestinal tract of a person knownto an ‘equol producer’ can convert, as they do in vivo, the glyconedaidzein to the final product S-equol.

Typical bacterial strains that can convert a glycone to an aglycon (suchas daidzein to daidzein) include Enterococcus faecalis, a Lactobacillusplantarum. Listeria welshimeri, a mixed culture of organisms isolatedfrom the intestinal tract of an ‘equol producing’ mammal. Bacteroidesfragilis, Bifidobacterium lactis, Eubactria limosum, Lactobacilluscasei, Lactobacillus acidophilous, Lactobacillus delbrueckii,Lactobacillus paracasei, Listeria monocytogenes, Micrococcus luteus,Proprionobacterium freudenreichii and Sacharomyces boulardii, andmixtures thereof.

Typical bacterial strains that can convert an aglycon to equol (such asdaidzein to S-equol) include Proprionobacteria freundenreichii, a mixedculture containing: Bifidobacterium lactis, Lactobacillus acidophilus,Lactococcus lactis, Enterococcus faecium, Lactobacillus casei andLactobacillus salivarius; and a mixed culture of organisms isolated fromthe intestinal tract of an ‘equol producing’ mammal.

The time required for bacterial conversion of the glucosides toaglycons, or the aglycons to the equol product, will depend uponbacteria-related factors, particularly concentration, the availabilityof oxygen, and the temperature and pH of the incubating system. In mostinstances it is possible to achieve substantially complete conversionwithin 24 hours.

The pH range for bacterial conversion of the isoflavone glucosides toaglycon isoflavones is from about 3 to about 9. The optimum pH dependsprimarily upon the type of bacteria used, and should be selectedaccordingly.

The time required for enzymatic conversion of the glucosides toaglycons, and aglycons to the equol product, depends upon enzyme-relatedfactors, particularly concentration, and the temperature and pH of thesystem. In most instances it is possible to achieve substantiallycomplete conversion within 24 hours, more preferably within about 2hours, and most preferably within about 1 hour.

S-equol produced in bulk can be separated from the resulting bulksolution of a bacterial production of S-equol, by methods well known inthe art, including crystallization, solvent extraction, distillation,and precipitation/filtration. The resulting bulk solution can containunreacted daidzein or other related isoflavone used, by-products, andany reactants. Such methods can include the use of a reverse-phase orstraight-phase liquid chromatography column and these can be combinedwith chiral-phase chromatography

A typical method of removing S-equol from a bulk solution or solid phaseis by extraction. An extractant solution is added to the solution orsolid phase containing the S-equol. Typically the extractant is a lowmolecular weight alcohol such as methanol, ethanol, isopropyl alcohol,or propyl alcohol, or an aqueous solution having a pH in the range from3.5 to 5.5. Typically, if the aqueous alcohol method is being used,sufficient alcohol is added to bring the alcohol/water ratio to betweena minimum of 40:60 and a maximum of 95:5. More typically, the ratio isat least 60:40, and even more typically a ratio between 65:35 and 90:10.

If an aqueous acid extraction method is used an aqueous acid solution isprepared with the pH adjusted to about 3.5 to about 5.5, and morepreferably within the pH range of about 4.0 to about 5.0. Sufficientwater is added to make a dilute liquid with a sufficiently low viscosityto permit separation of solids from liquids by centrifugation orfiltration.

The liquid, from which insoluble solid matter has been removed, isconcentrated by conventional methods for removing liquids. Methods usedtypically include, but are not limited to, removal of solvent byevaporation, preferably under reduced pressure. The residual liquid isconcentrated to at least about 15% solids, and up to about 55% solids,more typically to between 30% and 50% solids. The concentrate is thendiluted with water to reduce the solids content and increase the waterto alcohol ratio. The amount of water added can be varied over a widerange, though a final solids content between 6% and 15%, and moretypically about 13%, is preferred. The pH of the mixture is adjustedbetween about pH 3.0 and about pH 6.5, with a preferred value betweenabout pH 4.0 and about pH 5.0. Typically the temperature is betweenabout 2° C. to about 10° C., and more typically about 5° C. to 7° C.

The solid material is then separated from the liquid by standardseparation techniques (centrifugation or filtration) and yields anequol-rich solid material.

The equol-rich material can optionally be purified, typically bychromatography employing a silica gel column, with an eluent comprisingC2-C4 alcohols, C3-C7 alkanes, and mixtures thereof. The purifiedresidue can be crystallized from n-hexane to produce S-equol as a pureproduct, typically of at least 99%, with a yield typically of at least75%. The equol crystallized product is colorless, not hygroscopic, andstable in air, and does not decompose during the final filtrationprocedure.

The S-equol product can be authenticated by GC-MS analysis of thetrimethylsilyl ether or tert-butyldimethylsilyl ether derivative, orsome other appropriate volatile derivative of synthesized product as asingle pure peak and a mass spectrum that is consistent with thepublished electron ionization spectrum of the trimethylsilyl (TMS) etherderivative of authentic equol. Confirmation of the product can also beestablished by direct mass spectrometry using electrospray ionizationafter introducing the sample into the instrument via an HPLCchiral-phase column.

Treatment of Disease by Administering S-Equol. R-Equol, and Mixtures

This present invention provides a means for an individual subject toovercome the problem of not being able to produce equol in vivo, or tosupply R-equol in particular, by providing delivery of equolenantiomers, the S-equol or R-equol, or non-racemic mixtures of S-equoland R-equol directly, circumventing the need for intestinal bacteria forits production or for the need to consume soy foods with equal'sprecursor isoflavones. The delivery of S-equol can also supplement thein vivo production of S-equol in ‘equol-producers’, as well as in‘non-equol producers’.

Supplementing the diet of an equal producer with an equol enantiomer ormixture, can provide benefits when the ordinary level of S-equolproduced by the equol producer is inadequate because of 1) insufficientconsumption of isoflavones to produce equol, 2) antibiotic use thatablates the activity of intestinal bacteria to make equal from precursorisoflavones, or 3) other health factors that impact the level of equalproduction, e.g. short bowel syndrome or surgical construction of anintestinal stoma such as ileostomy. In addition, a supplemental level ofequol is believed to provide enhanced effect on the health andwell-being of the person.

This invention provides a method for delivering S-equol, R-equol,racemic equol, or non-racemic mixtures of equol, in sufficient amountsto have health benefits toward androgen-related diseases and conditionsassociated therewith. The anti-androgenic activity of equol can affect anumber of tissues throughout the body. In particular, the blocking ofandrogenic activity of DHT can be beneficial for the treatment andprevention of: (A) growth of the prostate gland with aging, benignprostatic hyperplasia (BPH) and prostate cancer; (B) female- andmale-pattern baldness, (C) facial and body hair growth (hirsutism), skinhealth (acne, anti-aging and anti-photo aging), skin integrity (collagenand elastin robustness); (D) body weight gain (and loss), reduction inadipose tissue deposition and metabolism of lipids, as well as generalregulatory behaviors and effects, such as food and water intake, bloodpressure changes, thyroid, glucose, leptin, insulin and the influence onthe immune system; and (E) Alzheimer's disease and emotional, mentalhealth issues, such as, mood, depression, anxiety and learning andmemory by reducing the 5α-steroid metabolites (covering androgens andprogesterone) that are potent modulators of the GABA_(A) receptor in thebrain that influences all of the brain characteristics above.

Typically, the amount of composition comprising the equol isadministered in an amount sufficient to produce a transient level ofenantiomeric equol in the blood plasma of the mammal of at least 5nanograms per milliliter (ng/mL), more typically at least 10 ng/mL orgreater, or transient levels of enantiomeric equol in urine of greaterthan 1000 nmol/L. Typically, the composition is administered orally in adose amount of at least about 1 mg, more typically of at least 5 mg, andof up to 200 mg, more typically, up to 50 mg, of enantiomeric equol. Atypical level of bioavailability of R-Equol in plasma after oraladministration of 20 mg of R-equol enantiomer to a healthy adult isshown in the appearance/disappearance plots of R-equol in FIG. 2.

The ability to deliver R- and/or S-equol in sufficient amounts isbelieved to provide several advantages over delivery of a racemicmixture of equol. First, the potency of R-equol or S-equol alone wouldtypically be at least twice that of the racemic mixture. Second, thehuman body only produces the S-equol, and therefore, a compositioncomprising only S-equol represents a “natural” product with aningredient, S-equol, with which the body is familiar. Third, since theR-equol enantiomer has unique properties, a treatment compositioncomprising only, or substantially only, the R-enantiomer can producebeneficial and/or therapeutic effects. And fourth, administration ofR-equol would supplement any endogenous S-equol present and allow forboth estrogenic and anti-androgenic actions to occur in the body.

The invention includes the use of enantiomeric equol to treat andprevent diseases and conditions related to male- and female-patternbaldness. DHT is a known cause of scalp hair loss. An androgen,specifically the principal circulating androgen, testosterone, isconverted to the more potent androgen, dihydrotestosterone (DHT) (in thehair follicle), and the hormonal action of DHT on scalp hair folliclescause hair loss. Thus, if the hormonal action of DHT can be blocked,such as by the use in the present invention of equol to bind DHT in thecirculation (within blood vessels) and within the hair follicle], thenscalp hair loss can be decreased or prevented.

The invention includes the use of enantiomeric equol to treat andprevent diseases and conditions related facial and body hair. Facial andbody hair are regulated by androgens, but oppositely to that of theregulation of scalp hair. Specifically, the more potent androgen, DHT,increases facial and body hair. DHT also increases the production ofsebum (oil) from the sebaceous gland, which can contribute to anincrease in acne. Thus, the binding of DHT by equol can cause a decreasein facial and body hair and in secretion of sebum (oil), and a reductionor prevention of acne.

The invention includes the use of enantiomeric equol to treat andprevent diseases and conditions related to skin effects, skin qualityand integrity, skin aging, skin photoaging, and skin pigmentation andlightening. Estrogens, before but especially after menopause, improveskin health by increasing elastin and collagen content to improve skincharacteristics or robustness. Also, when skin is damaged by acne orother skin disruptions (scratches, popping pimples or minor cuts, etc.),the repair mechanism is faster and the skin heals better if estrogen orestrogen-like compounds, such as equol, are present. It is believed thatan equol enantiomer mixture, and particularly S-equol, is a goodstimulator of elastin and collagen and also can protect againstphoto-aging. Equol's blocking the hormone action of DHT can decreasesebum oil production from the sebaceous gland, which can decrease oreliminate acne. Since S-equol (though not R-equol) binds estrogenreceptor(s) (mainly ERβ), the protective effects of this estrogen-likemolecule would stimulate elastin and collagen in the skin. Additionally,since equol is a strong antioxidant, it can protect the skin from aging,including photo-aging.

The invention includes the use of enantiomeric equol to treat andprevent diseases and conditions related to improved prostate health. Theconversion of testosterone to the more potent androgen DHT is the resultof the action of the enzyme 5α-reductase within the prostate. DHT causesbenign prostatic hyperplasia (BPH), increases in the prostate weight,and can result in the need for prostatectomies and radiotherapy to treatthese conditions. Finally, consumption of soy foods has receivedincreased attention due to their ‘health benefits’ of decreasing hormonedependent cancers such as prostate and breast cancer. Thus, blockage ofDHT by equol decreases prostate weight in animal models and presumablywill block BPH to prevent prostate cancer.

The invention includes the use of enantiomeric equol to treat andprevent diseases and conditions related to brain function and mentalhealth, including brain disorders, dementia of the Alzheimer type, aswell as other reduced or impaired cognitive functions associated withadvancing age and with short- and long-term memory loss. Brainmechanisms are more complex, and attempting to define what molecules andfactors regulate, influence, etc., mood, depression, anxiety and so on,can be difficult. However, there are some data to support the conceptthat estrogens or estrogen-like molecules like isoflavones can assistcognitive function in conditions such as Alzheimer's disease, and mayhelp to prevent the onset of such disorders, especially inpostmenopausal women.

In reference to mood, anxiety, depression, and other mental healthconditions, there are two basic viewpoints. One line of researchsupports the view that estrogens (especially in women) regulate anxietyand help to decrease anxiety levels. Both estradiol and progesteronealter anxiety-related behaviors as well as the testicular androgen,testosterone (Imhof J. T. et al, Behav Brain Res, 56:177-180 (1993). Theanti-androgenic activity of equol acts in the brain by enhancingneurotransmission and restoring synaptic density. Without being bound byany particular theory, we believe that R- and/or S-equol are active inthe brain at the same site(s) as estrogen, exerting an estrogenicresponse.

The second line of research is more complex and supports the view that5α-reduced steroids, especially progesterone, have the ability to bindGABA_(A) receptors in the brain and cause sedation. GABA_(A) is themajor inhibitory neurotransmitter in the brain and its receptors areabundant in brain areas that control mood/emotion. By causing sedation,individuals express less anxiety. For example, most women duringpregnancy, report that they feel OK but they are usually tired orsleepy. This is due to progesterone being converted by the 5α-reductaseenzyme in the body, but especially in the brain to5α-dihydroprogesterone (5α DHP) and further metabolism of this moleculeresults in the most potent ‘neurosteroid’ that can bind the GABA_(A)receptor and enhance the action of GABA_(A). Additional supportingevidence that this 5α-dihydroprogesterone molecule (and its metabolite)can decrease brain activity is seen in epileptic women (who experienceepilepsy and hence seizures) where these individuals almost neverexperience seizures during pregnancy due to the high circulating levelsof progesterone. It should be noted that 5α-reduced androgens like5α-DHT also have a similar effect on GABA_(A) receptors to causesedation (in men) but at much lower levels compared to 5α-DHP.

Putting these two views together, estrogen on the one hand decreasesanxiety and hence increases activity. Conversely, blocking the action of5α-DHP also increases activity and thus in behavioral tests isinterpreted as decreasing anxiety. For example, when the conversion ofprogesterone to 5α-DHP in pregnant rats has been blocked, this resultsin a significant increase in their locomotor activity levels.

Taking a similar perspective on this in reference to equol, equol hasthe ability to bind 5α-DHP (mainly seen in women) and 5α-DHT (mainlyseen in men). This would decrease the potent ‘neurosteroid’ effects atthe GABA_(A) receptor and decrease sedation and thus increase activityor decrease anxiety. Moreover, the ability of S-equol to bind theestrogen receptor(s) beta would also increase activity. Finally, ourstudies using young- or mid-aged adult rats, in males or females haveshown that dietary phytoestrogen consumption (Lund T. D. et al. BrainRes, 913:180-184 (2001); Lephart, E. D. et al, NeurotoxicologyTeratology, 24: 1-12 (2002)), or injections with equol significantlydecrease anxiety levels as expressed in the elevated plus maze test.

One report, conversely, suggests that isoflavones can increase anxietyin male rats (Hartley et al., Psychopharmacology, 2003, 167:46-53).

The elevated plus maze is a behavioral test used to quantifyanxiety-related behavior and identify anxiolytic drugs (Pellow S. et al,J Neurosci Methods, 14:149-147 (1985); Current Protocols In Neuroscience(1997) 8.3.1-8.3.15, John Wiley & Sons. NY, NY). The test relies on theinherent conflict between exploration of a novel environment andavoidance of its aversive features. Normally, animals spend little timeand make few entries into the open arms of the maze compared to theclosed arms of the maze (Imhof J. T. et al., Behav Brain Res, 56:177-180(1993). However, animals treated with anxiolytics, such asbenzodiazepines (valium), spend more time in the open arms and thenumber of entries into the open arms reflects a decrease inanxiety-related behaviors (Pellow S. et al, J Neurosci Methods,14:149-147 (1985); Current Protocols In Neuroscience (1997)8.3.1-8.3.15. John Wiley & Sons. NY, NY; Chopin P. et al., Psychopharm,110:409-414 (1993).

The invention includes the use of enantiomeric equol to treat andprevent diseases and conditions related to body weight and body fatformation. Phytoestogens including equol, have the ability to decreasethe formation of white adipose (fat) tissue and increase white adiposetissue breakdown, thus decreasing body weight. Also, the estrogen-likenature of phytoestrogen molecules decreases LDL (so-called “bad”cholesterol), blood pressure, and prevents insulin resistance (or inother words, provides beneficial effects to the diabetic condition).Since equol is a more potent isoflavone molecule compared to the otherphytoestrogens, it presumably provides the health benefits and protectsagainst the conditions outlined above.

Since equol binds DHT, equol can also block the actions of DHT thatpromote body weight gain. Thus, equol, and particularly S-equol,combined anti-androgenic but at the same time an estrogenic hormoneaction from the same molecule (equol) would further improve the healthbenefits of body weight loss (and weight management), decrease LDLcholesterol, decrease blood pressure, and help prevent the devastatingeffects of diabetes.

The invention includes the use of equol, as enantiomeric equol ormixture thereof, to treat and prevent lipid disorders such as highcholesterol (hypercholesterolemia), lipidemia, lipemia and dyslipidemia(disturbances in lipids). A study has shown that plasma totalcholesterol concentrations decreased 7.2% (p=0.04) in equol producerscompared with baseline levels and 3.0% (p=NS) in non-equol producers.The failure of soy protein to have significant cholesterol-loweringeffects in adults with normal blood cholesterol levels, is, with fewexceptions, probably because of heterogeneity in the study populationswith regard to the metabolism of soy isoflavones and the failure torecognize the relevance of equol formation (and specifically,non-formation in non-equol producers). These data suggest thatenantiomeric equol influences lipids in a favorable manner, and that theeffect is mediated by androgens. The composition comprising equol isadministered in an amount sufficient to reduce the level of lipids inthe blood stream.

The invention further includes the use of R- and/or S-equol to improvediminished blood vessel quality, by increasing reactivity or flexibilityin response to acute changes in blood pressure, improving blood flow,and reducing blood pressure. The invention also includes the use of R-and/or S-equol to treat and prevent cancer, including benign prostatecancer, prostate cancer, and skin cancer.

Another embodiment of the present invention is the use of equol to treatenlarged prostate or epididymis. Equol may also be used to preventenlarged prostate or epididymis in individuals believed to be at riskfor development of these pathologies, without alterations in testes,pituitary or body weights. The equal may be administered by any routethat allows absorption of equal to the blood stream.

DHT-Androgen Receptor

Other embodiments of the present invention include the use of equol as adiagnostic agent in androgen-related disorders as well as disordersarising from disturbances in estrogenic/androgenic balance. In theseembodiments, equol is administered to an individual to bind DHT andthereby prevent DHT binding to androgen receptors. The changes inestrogenic balance are then measured or the change in androgen-bindingis assessed to diagnose or further elucidate androgen-related anomalies.

Binding to DHT

Equol can be administered to bind DHT prior to or along with othertherapeutic moieties in order to assess the binding capacity of DHT withrespect to the therapeutic moiety in question. Also, androgen-bindingmoieties can be administered following administration of equol to assessthe efficacy of the androgen-binding moiety to restore androgen activityand balance estrogenic activity in the absence of DHT binding. Further,equol can be administered in the presence of DHT-binding moieties inorder to displace these naturally-occurring or xenobiotic DHT-bindingmoieties from DHT.

Administration of Equol

In each of the embodiments of the present invention described herein, ifthe administration of equol is to be oral, the equol may be administeredby supplying an oral dosage form of equol to either an “equol-producing”mammal or a “non-equol producing” mammal, or an oral dosage of daidzein,daidzin, isoflavone mixtures containing daidzein, or soy proteinpreparations to an “equol-producing” mammal, wherein the administrationof the oral dosage form results in effective absorption of equol to theblood stream. Administration of equol may be made by routes other thanoral if desired. For example, it is contemplated that rectal or urethraladministration may be used to administer equol for the treatment ofenlarged prostate or to prevent prostate enlargement. Additionally, itis contemplated that the active ligand binding site of the equolmolecule may be isolated and synthesized for administration, which canprovide DHT binding without the full equol molecule. The dose of theequol molecule or fragment thereof having DHT-binding abilities isdependent upon the route of administration and the condition to betreated. Based on our in vivo studies it is apparent that relatively lowdoses of equol antagonize much higher doses of DHT, and this may beexplained by the marked differences in the binding of equol to serumprotein compared with DHT. The latter circulates mostly bound toproteins, while equol is 50% free. Generally, a dose sufficient toproduce a concentration of equol or active fragments thereof in thebloodstream of the recipient of at least about 0.2 mg equol per kgweight of the recipient and preferably at least about 0.5 mg/kg. Thedose may be increased dramatically without incurring significantdose-limiting side effects to greater than about 10 mg/kg. Oraladministration can be effected in microencapsulated forms that canprovide delayed or sustained release of the medicament.

Equol can be administered topically, transdermally, and subdermally in avariety of forms, including lotions, ointments, foams (including shavingcreams), nasal sprays, skin patches (such as described in U.S. Pat. No.5,613,958, incorporated herein by reference), electromechanical devices,including micropumps systems (such as described in U.S. Pat. No.5,693,018 and U.S. Pat. No. 5,848,991, incorporated herein byreference), and subdermal implants (such as described in U.S. Pat. No.5,468,501, incorporated herein by reference).

Experiments (a) Determination of Equol Enantiomer in ‘Equol-Producing’Adults

The urine samples from adults consuming soy foods previously identifiedas being ‘equol-producers’ were analyzed. Equol was isolated from urine(25 mL) by passage of the sample through a solid-phase Bond Elut C18cartridge. After washing the cartridge with water, the isoflavones wererecovered by elution with methanol (5 mL) and the methanolic phase wastaken to dryness under a stream of nitrogen. The sample was subjected toenzymatic hydrolysis with Helix pomatia and then re-extracted on a BondElut C18 cartridge. The methanolic extract was taken to dryness undernitrogen gas and redissolved in HPLC mobile phase (100 μL). Equolenantiomers were identified by HPLC using a Chiralcel OJ chiral phasecolumn as described herein above. The detection of equol was achieved byselected ion monitoring electrospray ionization mass spectrometry(ESI-MS). Mass chromatograms of a pure standard of S-equol, and of urinefrom an adult consuming soy food are shown in FIG. 3. Similar studieshave demonstrated that soy-derived isoflavones are converted to equol inrats, as well, thus validating rodent models of isoflavone metabolism.

The retention index and mass chromatogram establish that it isexclusively the S-enantiomer of equol that is excreted in human urine asno detectable R-enantiomer of equol could be found. Analysis of theplasma from the same ‘equol-producer’ also revealed only theS-enantiomer of equol.

(b) Chemical Synthesis of Racemic Equol

Daidzein (200 mg, 0.8 mmol) is dissolved in a mixture of glacial aceticacid (20 mL) and isopropanol (20 mL), and is reduced with 10% Pd oncharcoal (150 mg) at 55 p.s.i.g. (3.7 atm gauge). At the end of thereaction (2 hours, TLC:isopropanol/n-hexane 1/4) the catalyst isfiltered off, and the filtrate is evaporated. The crude residue ispurified by chromatography on a silica gel column using as eluent amixture of isopropanol and n-hexane (1:4 v/v), to give (±)equol as apure product (160 mg, yield: 82%) crystallized from n-hexane. Theproduct, colorless crystals, is not hygroscopic, is stable in air, anddoes not decompose during the final filtration procedure. The product ofthis chemical synthesis was in all respects identical with an authenticsample of (±)equol (racemic equol). FIG. 4 shows the GC-MS analysis ofthe trimethylsilyl ether derivative of synthesized product as a singlepure peak and a mass spectrum that is consistent with the publishedelectron ionization spectrum of the trimethylsilyl (TMS) etherderivative of authentic equol. The molecular ion as expected is at m/z470 and the base peak at m/z 234. The purified equol product had apurity of greater than 99%, as confirmed by HPLC and mass spectrometry.

(c) Elution Order of S- and R-Enantiomer by Optical Dichroism

A racemic mixture of S-equol and R-equol were separated by chiralchromatography on a Chiralcel OJ Column using a flow-rate of 1.0 mL/minand with a gradient elution consisting of an initial mobile phase of 10%ethanol in hexane and increasing to 90% ethanol in hexane over a timeperiod of 15 minutes according to the program shown in Table 1:

TABLE 1 Chiral separation gradient eluent of hexane and ethanol Time(min.) % hexane % ethanol 0 90 10 1.0 90 10 15.0 10 90 16.0 90 10 17.090 10

FIG. 5 shows the mass chromatogram of the ions recording (m/z 241) for aracemic mixture of S- and R-equol.

The first eluting material, designated as Enantiomer-1, and the secondeluting material, designated as Enantiomer-2, were collected separately.Each enantiomer was weighed and the weighed samples dissolved in 1 mL ofspectroscopic grade ethanol. Measurement of the optical rotation of eachenantiomer was carried out at 20° C. using the light of wavelength inthe line D of sodium.

Enantiomer-1 material (1.6 mg exact weight) had first and secondmeasurements of −0.023 and −0.022, resulting in an optical rotation of−14 [−0.0225×1000/1.6], which corresponds with the S-enantiomer ofequol. Enantiomer-2 material (1.7 mg exact weight) had first and secondmeasurements of +0.023 and +0.023, resulting in an optical rotation of+13.5[+0.023×1000/1.7], which corresponds with the R-enantiomer ofequol.

d) Determination of Receptor Binding Capacity of S- and R-Enantiomers

In vitro binding studies were performed to examine the relativeaffinities of S- and R-enantiomeric equol with the estrogen receptorsERα and ERβ.

Synthesis of Hormone Receptor Proteins: Full length rat ERα expressionvector (pcDNA-ERα: RH Price UCSF) and ERβ expression vector (pcDNA-ERβ;TA Brown. Pfizer. Groton, Conn.) were used to synthesize hormonereceptors in vitro using the TnT-coupled reticulocyte lysate system(Promega, Madison, Wis.) with T7-RNA polymerase, during a 90 minreaction at 30° C. Translation reaction mixtures were stored at −80° C.until further use.

Saturation isotherms: In order to calculate and establish the bindingaffinity of the S-equol and R-equol enantiomers for ERα and ERβ, 100 μLaliquots of reticulocyte lysate supernatant were incubated at optimaltime and temperature: 90 min at room temperature (ERβ) and 18 hrs at 4°C. (ERα), with increasing (0.01-100 nm) concentrations of[3H]17β-estradiol (E2). These times were determined empirically andrepresent optimal binding of receptor with estrogen. Nonspecific bindingwas assessed using a 300-fold excess of the ER agonist,diethylstilbestrol, in parallel tubes. Following incubation, bound andunbound [3H]E2 were separated by passing the incubation reaction througha 1 mL lipophilic Sephadex LH-20 (Sigma-Aldrich Co., Saint Louis, Mo.)column. Columns were constructed by packing a disposable pipette tip (1mL; Labcraft, Curtin Matheson Scientific, Inc, Houston, Tex.) with TEGMD(10 mm Tris-Cl, 1.5 mm EDTA, 10% glycerol, 25 mm molybdate, and 1 mmdithiothreitol, pH 7.4)-saturated Sephadex according to previouslypublished protocols (Handa et al., 1986: O'Keefe and Handa, 1990). Forchromatography, the columns were re-equilibrated with TEGMD (100 μL),and the incubation reactions were added individually to each column andallowed to incubate on the column for an additional 30 min. Followingthis incubation, 600 μL of TEGMD were added to each column, flow-throughwas collected, 4 mL scintillation fluid was added, and samples werecounted (5 min each) in an 2900 TR Packard scintillation counter(Packard Bioscience, Meriden, Conn.).

Competition binding studies were used to assess the estrogenicproperties of equol's S-equol and R-equol enantiomers. Based on theability of S and R to compete with [3H]E2 for ER binding, the affinitiesfor in vitro translated ER were shown to be very different for the twoenantiomers. The S-equol enantiomer showed greatest affinity for ERβ [Kd(nm)=0.73±0.2], while its affinity for ERα was relatively low bycomparison [Kd(nm)=6.41±1.0]. The R-equol enantiomer possessed a muchlower affinity for both ERβ [Kd (nm)=: 15.4±1.3] and ERα [Kd(nm)=27.38±3.8]. For reference 17β-estradiol binds ERα with a Kd(nm)=0.13 and ERβ with a Kd (nm)=0.15 in this system.

The study shows that only the S-equol enantiomer binds ER withsufficient affinity to have potential relevance to circulating equollevels reported in humans. Compared with 17β-estradiol the relativebinding affinities of the S-equol and R-equol enantiomers for ERα were49-fold and 211-fold less, respectively. However, the S-equol enantiomerseems to be largely ERβ-selective with a relatively high affinity forERβ, while the R-equol enantiomer binds with approximately 100-fold lessaffinity. The separate and associated determination that exclusivelyS-equol is found in human plasma and urine is significant in view of thespecificity in binding of the two enantiomers.

e) Bioavailability of R-Equol

20 mg of pure R-equol was administered orally to a healthy adult afteran overnight fast. Blood samples were collected at timed intervals overthe next 24 hours and the plasma concentration of equol was determinedby isotope dilution gas chromatography-mass spectrometry with selectedion monitoring. Rapid appearance of equol is observed in the plasma withpeak concentrations observed after 8 hours. The terminal eliminationhalf-life of R-equol was approximately 8 hours. Electrospray ionizationmass spectrometry confirmed that the equol present in plasma was theR-equol enantiomer (data not shown but available on request), therebyestablishing that it is stable and does not undergo any racemization orfurther biotransformation in the intestine. FIG. 2 shows anappearance/disappearance plot of R-equol. These results establish thatR-equol if administered as a pharmacologic or nutraceutical preparationis extremely bioavailable.

EXAMPLES

Where appropriate, data were analyzed by analysis of variance statistics(ANOVA) followed by Newman-Keuls post hoc tests. Significance was set atp<0.05. Curve fitting, scientific graphing, and analysis were completedusing GraphPad Software (GraphPad Prism 3.0, San Diego, Calif.).

Example 1

This example demonstrates the in vivo effects of equol on prostate sizeand hormone secretion. Male Sprague-Dawley rats (400-500 grams) areobtained from Charles Rivers Laboratories (Wilmington, Mass., USA). Ratsare caged in pairs and maintained on a 12-hour dark 12-hour lightschedule (lights on at 0700 h) with ad libitum access to food and water.

One week following arrival, animals are given subcutaneous (sc)injections (1/day for 4 days) of either dimethylsulfoxide (DMSO)(vehicle control) or racemic equol (0.25 mg/kg). Eighteen hours afterthe final injection, animals are killed via decapitation and trunk bloodand prostate are collected for analysis.

A significant reduction in prostate weight is observed in intact malesinjected subcutaneously with equol in comparison to intact controlmales. Additionally, luteinizing hormone (LH) in these same intact malesis significantly increased in equol compared to control treated males.These finding are shown in FIGS. 6 A and 6B, respectively. These effectsare observed with relatively low levels of equol compared to DHT andthis can be explained by the marked differences in the protein bindingof equol, which circulates about 50% free, and DHT which is mostly boundto serum protein.

Example 2

In addition to equol's effects on prostate racemic equol blocks theeffects of DHT in other tissues, and decreases body weight. One weekfollowing arrival, intact males are given subcutaneous injections ofeither DMSO (control) or equol (0.5 mg/kg) once/day for 7 days.Following treatments animals are weighed and then killed viadecapitation and tissue collection (prostate, testes, epididymis, andpituitary).

A significant weight decrease in the DHT-sensitive epididymis isobserved in racemic equol-treated males compared to controls. However,racemic equol did not affect testes weight or pituitary weight. Bodyweight during this relatively brief treatment period does not differsignificantly between racemic equol and control treatments. Theseresults are presented in Table 2.

TABLE 2 Tissue weights from intact males given subcutaneously injectionsof either DMSO (control) or racemic equol. Prostate Epididymis TestesPituitary Body (g) (g) (g) (g) (g) DMSO 0.58 1.91 3.52 0.011 428.2(Control) (±0.05) (±0.10) (±0.10) (±0.003) (±4.04) Racemic Equol 0.381.59 3.48 0.014 418.0 (0.25 mg/kg) (±0.06)* (±0.06)* (±0.08) (±0.002)(±7.52)

Example 3

Adult male Sprague-Dawley rats are randomly assigned to three groups andreceive daily injections of either DMSO, a racemic mixture of equol at0.250 mg/kg/day), R-equol at 0.250 mg/kg/day, or S-equol at 0.250mg/kg/day. The total volume of each injection is 0.3 cc, administered scat the nape of the rat's neck. After seven consecutive days oftreatment, the rats are killed and the body weight gain during theinjection period is determined. Rats injected with R-equol have asignificant decrease in body weight gain compared to control rats, asshown in Table 3.

TABLE 3 Body Weight Gained in Male Sprague-Dawley Rats Treated withEquol. Body Wt Gain Treatment Group Prostate (g) Epididymis (g) (g)Control 0.38 (±0.01) 0.96 (±0.03) 59.4 (±3.4) Racemic Equol 0.35 (±0.02)0.89 (±.03)  56.1 (±3.7) (0.25 mg/kg) R-equol (0.25 mg/kg)  0.31(±0.02)*  0.85 (±0.04)*  45.6 (±5.5)* S-equol (0.25 mg/kg) 0.35 (±0.03)0.86 (±0.05) 56.1 (±3.1) *Significant reduction compared to control

Testis and pituitary gland weights are not significantly altered by thetreatments (data not shown). The slight decreases in body weight (around10%) from the equol experiments are very similar to those seen betweenanimals fed a Phyto-Free diet (a diet containing very low levels ofphytoestrogens) vs. a Phyto-600 diet (a phytoestrogen-rich dietcontaining 600 ppm of isoflavones). The significant reduction in whiteadipose tissue deposition with the racemic equol injections (around 36%)is also comparable with that seen with the data sets derived from thedietary treatment studies. These findings suggest that R-equol canregulate body weight and significantly decrease white adiposedeposition.

Example 4

This Example demonstrates equol binding to DHT. In initial bindingcompetition studies conducted to determine and establish equol's bindingaffinity for AR, we repeatedly observed that the apparent binding of[3H]DHT was greater in the presence of equol than in its absence. Slightmodifications in the protocol where AR was removed from the incubationtube (leaving only [3H]DHT and equol) resulted in the elution of [3H]DHTinto the eluate containing [3H]DHT reaction complex.

To further investigate this phenomenon, a 30 cm long Sephadex LH-20columns are used in order to identify elution peaks establishing thebinding of [3H]DHT to equol. As shown in FIG. 7, a peak of [3H]DHT isapparent in the elution fractions between 5 and 9 mL when the[3H]DHT+equol column incubate is applied. This peak is not present when[3H]DHT alone is applied to the column. Furthermore, when DHT orDHT+equol are incubated with prostate supernatant and then passedthrough the 30 cm column (FIG. 8A) two distinct binding peaks areidentifiable. The first peak of [3H]DHT represents that bound to the ARin prostate. This is found in the elution fractions between 4 and 5 ml.In addition there is a later peak (between 5 and 9 ml), consistent withthe binding of [3H]DHT to equol. However, when [3H]DHT is allowed toincubate with the prostate supernatant for 36 hours (until equilibrium)prior to the introduction of equol there is no apparent binding of[3H]DHT (FIG. 8B). Both [3H]DHT and [3H]DHT+equol (equol added 36 hourslater) show a single peak in the elution between 4 and 5 ml, suggestingthat equol does not compete with DHT for the AR nor does it bind [3H]DHTthat is already bound to the receptor. Furthermore, it should be notedthat the binding of equol to DHT appears to be specific, since similarcompetition and binding studies have been conducted using [3H]E2, [3H]T,[3H]DHEA, [3H]CORT and [3H]progesterone without any occurrences ofbinding to equol (data not shown). Saturation analysis of equol bindingto [³H]DHT shows an apparent Kd calculated at 1.32±0.4 nM (FIG. 9).

Example 5

This example demonstrates that, in addition to modulating the effect ofDHT on prostate size, equol binds to DHT in vivo and blocks the negativeeffects on LH secretion. GDX males treated with a long-lasting analog ofDHT, DHT propionate (DHTP), show a significant increase in prostateweight compared to vehicle-treated GDX control rats. Concomitanttreatment with equol (DHTP+equol) blocks the effects of DHTP, whileequol has no effect alone on prostate size as shown in FIG. 10A. Equolalso blocks DHT's negative feedback effects on LH. In GDX males LH issignificantly decreased by DHTP treatment compared to treatment withDMSO. Treatment with equol in combination with DHT blocks the negativefeedback effects of DHTP on LH secretion. Equol alone has no effect onLH levels, shown in FIG. 10B.

Example 6

This example demonstrates the effects of racemic equol onandrogen-sensitive tissues. One week following arrival, animals aregonadectomized (GDX) under isoflurane anesthesia and allowed to recoverfor 7 days. Following recovery, animals are assigned to the followinggroups 1) DMSO, 2) DHTP (2 mg/kg), 3) racemic equol (0.25 mg/kg), or 4)both DHTP and racemic equol. Injections are given subcutaneously dailyfor 4 days. Animals are killed via decapitation and trunk blood andtissues are collected for analysis. Plasma DHT is measured, shown inFIG. 11. As expected there were significant elevations of plasma DHT inanimals treated with DHTP (GDX+DHTP, GDX+equol+DHTP groups). Plasma DHTwas further elevated, although, not significantly, by co-treatment withequol. Tissues, including prostate, testes and epididymis, are removedfrom the animal, dissected free of fat and connective tissues, weighed,fixed by immersion in 4% paraformaldehyde, and then sectioned at 15 μmon a cryostat. Tissue sections are mounted on charged slides (SuperfrostPlus. Fisher Scientific, Pittsburgh, Pa.) prewarmed to 23° C., andstained with hematoxylin and eosin (H&E), dehydrated in ascendingalcohol and cleared with xylene. Histological sections are shown inFIGS. 12 and 13. H&E stained prostates reflect a change due to both GDXand treatments. The prostate glands of control, equol, and DHTP plusequol treated groups show similar histology (FIG. 12 A, B, D). In theseanimals prostates are characterized by very small atrophic glands withlittle volume in the gland lumen. In DHTP-treated animals (FIG. 12 C),the glands show signs of cell proliferation. Lumen size is increasedcompared to GDX animals; the epithelium is of a tall columnar type (FIG.12 C). In comparison to intact control animals (FIG. 12 E) the prostateof equol treated males show involution and consist of more closelyspaced, atrophic glands (FIG. 12 F). In comparison to control males, theepididymal histology of equol-treated intact males shows overall smallerducts, as evidenced by shrunken lumen (FIG. 13).

Example 7

Long-Evans male rats are raised (life long from conception to time ofsample collection) on either a phytoestrogen-rich diet containing 600micrograms of isoflavones per gram of diet or 00) ppm of isoflavones(referred to hereafter as the “Phyto-600” diet) or a diet containingvery low levels of isoflavones (referred to hereafter as the‘Phyto-Free’ diet; containing approximately 10 ppm of isoflavones). Asshown in FIG. 14 male Long-Evans rats fed the Phyto-600 diet displaysignificantly lower body weights at 33, 55 or 75 days of age compared toanimals fed the Phyto-Free diet. Adipose tissue (dissected from justbelow the kidneys to just above the testes in the abdominopelvic cavity)is measured in the 55 or 75 day-old males. At both ages, white adiposetissue mass is significantly greater in the Phyto-Free-fed malescompared to Phyto-600-fed animals, FIG. 15. It should be noted that thereductions in body weight of Phyto-600-fed males are modest, atapproximately 10 to 15% percent, whereas, the reductions in whiteadipose tissue from the same animals are approximately 50-60% comparedto Phyto-Free-fed males. This greater reduction in white adipose tissuecompared to body weight in soy fed animals is also a generalcharacteristic seen in humans consuming soy-based diets (D. B. Allisonet al, Eur J Clin Nutr, 2003, 57: 514-522. This particular result isrepeatedly seen throughout the various experiments present in these datasets, regardless of age, sex, rat strain or whether female rats havetheir ovaries removed (simulating the postmenopausal condition inhumans).

When food and water intake is measured to determine whether theseparameters might influence body and adipose tissue weights,Phyto-600-fed males display slight but significantly higher food FIG.16A) and water (FIG. 16B) intakes compared to Phyto-Free-fed animals.Thus, the reductions in body and adipose tissue weights cannot beexplained by alterations in food/water intake between the diettreatments.

Since leptin is produced by adipose tissue (D. A. Sandoval et al., JDiabetes Complications, 2003, 17:108-113.), serum leptin levels aremeasured, along with insulin levels, to determine the alterations inthese metabolic hormones. As shown in FIG. 17A, leptin levels at 33, 55or 75 days of age are significantly decreased in Phyto-600-fed males(which corresponds with the reductions seen in adipose tissue weightsfrom these same animals) compared to Phyto-Free-fed males. Also, insulinlevels are significantly decreased in Phyto-600-fed male vs.Phyto-Free-fed males (FIG. 17B), consistent with the benefits ofprotecting against insulin resistance associated with type-2 diabetes(V. Jayagopal et al., Diabetes Care, 2002, 25:1709-1714.).

To demonstrate that circulating isoflavone levels are different inPhyto-600-vs. Phyto-Free-fed male and female (75 day-old) rats, serumisoflavone levels are determined by GC/MS as previously performed by ourlaboratories (see methods in K. D. R. Setchell, Am J Clin Nutr129:1333S-1346S, 1998; and K. D. R. Setchell et al, J Nutr132:3577-3584, 2002.). In each case for the different classifications ofisoflavones Phyto-600-fed males display significantly higher isoflavonelevels compared to Phyto-Free-fed values, shown in Table 4. Moreimportantly, equol levels in the Phyto-600-fed rats account forapproximately 78% of the total phytoestrogen levels.

TABLE 4 Isoflavone concentrations in adult male and female rats TotalGenistein Daidzein Equol (ng/ml) Males: Very Low  9.6 ± 0.3 10.8 ± 0.6 23.2 ± 0.4 43.5 ± 1.0 Isoflavone Diet High 413 ± 67 394 ± 58  1,161 ±325  1,967 ± 45   Isoflavone Diet Females: Very Low  3.9 ± 0.2 5.3 ± 0.821.6 ± 1.2 30.8 ± 2.2 Isoflavone Diet High 99 ± 9 117 ± 7.4  931 ± 211,147 ± 5   Isoflavone Diet

To determine if other metabolic hormones were altered by the diettreatments or by age, serum glucose and thyroid (T3) levels are assayed.Glucose levels are slightly (but not significantly) higher in thePhyto-600-fed males compared to Phyto-Free-fed values, independent ofage or source of the blood samples [either arterial (ART) or venous(TRUNK)], shown in FIG. 18. However, when T3 levels are quantified,there is a significant increase in T3 serum levels in 80 or 110 day-oldmale Long-Evans rats fed the Phyto-600 diet compared to Phyto-Free-fedanimals, shown in FIG. 19. This suggests that thyroid levels areenhanced with soy consumption consistent with anecdotal evidence ofindividuals that decreased their thyroid medication or went off ofthyroid treatment completely with the consumption of soy based foods intheir diets. This is also consistent with reports of a similar increasein T3 levels in humans following consumption of soy foods (Watanabe, S.et al, Biofactors 2000: 12(1-4):233-41).

Example 8

In this experiment, female Long-Evans rats are raised (life long fromconception to time of sample collection) on either Phyto-600 orPhyto-Free diets. As shown in FIG. 20, rats fed the Phyto-600 dietdisplay significantly lower body weights at 80 days of age compared toanimals fed the Phyto-Free diet, representing about a 12% reduction inbody weight in the Phyto-600-fed animals. As seen in the male Long-EvansPhyto-600-fed rats previously, females fed the Phyto-600 diet alsodisplayed significant reductions in adipose tissue weight (by about 68%)compared to females fed the Phyto-Free diet, shown in FIG. 21.

Similar to results with male rats, serum glucose levels are slightly butnot significantly higher in Phyto-600-fed female rats at 80 or 110 daysof age compared to animals in the Phyto-Free diet treatment group, shownin FIG. 22. However, T3 levels are significantly higher in females fedthe Phyto-Phyto-600 diet compared to Phyto-Phyto-Free fed animals at 110days of age, shown in FIG. 23. The T3 and glucose results in females arevery similar to those obtained in male rats exposed to the same diettreatments, and thus, suggest similar health benefits for both genders.Samples collected at 100 days of age yield similar results (i.e.,significant reductions in body weight and adipose tissue weights withthe consumption of the Phyto-600 diet vs. the Phyto-Free diet) in femaleLong-Evans rats (data not shown).

Example 9

Adult female rats are placed on the Phyto-600 or Phyto-Free diettreatments from 50 to 215 days of age. Prior to 50 days of age theanimals can be raised on a diet that contains approximately Phyto-200ppm of isoflavones, or similar a diet such as those used by animalsuppliers. At 215 days of age. Phyto-600-fed females weigh significantlyless than Phyto-Free-fed females, representing about a 7% reduction inbody weight, shown in FIG. 24. White adipose tissue in Phyto-600-fedfemales at 215 days of age is significantly reduced by about 30%compared to that of females fed the Phyto-Free diet, shown in FIG. 25.Correspondingly, serum leptin levels in the Phyto-600-fed females weresignificantly lower than those of Phyto-Free-fed, shown in FIG. 26.Insulin levels were reduced in the Phyto-600-fed vs. Phyto-Free-fedfemales to a similar degree seen previously, but did not reachstatistical significance, shown in FIG. 27.

Example 10

This example demonstrates the effects of a Phyto-600 or Phyto-Free dieton adult ovariectomized (OVX) rats. The OVX rat is a well-establishedanimal model of postmenopausal human females. In addition, OVX permitsthe subcutaneous injections of estrogen and progesterone to stimulatebehavioral estrus in rats, to determine the effects of a Phyto-600 orPhyto-Free diet. Adult ovariectomized rats are fed a phytoestrogen dietof approximately 200 ppm of isoflavones (“Phyto-200”) until 50 days ofage (all animals are ovariectomized at approximately 40 days of age).The female rats are age and weight matched at 50 days of age and placedinto one of two diet treatments: either the Phyto-600 (black bars) orPhyto-Free (white bars) until 94 days of age. Baseline body weights aretaken at 50 days of age before the animals are placed on the diettreatments, again at 58 days (8 days of diet treatment), at 92 days ofage (before injection of estradiol), and at 94 days of age (beforeinjection of progesterone, and 6 hours later at 94 days of age (afterchemical induction of behavioral estrus), shown in FIG. 28.

After consuming the diets for 8 days, the Phyto-600-fed rats display aslight but significant reduction in body weight (of about 7%) comparedto Phyto-Free-fed. This reduction in body weight is maintained beforeand during the chemical induction of behavioral estrus by the estrogenand progesterone (steroid) injections.

White adipose tissue is measured at 94 days of age after the chemicalinduction of behavioral estrus, Phyto-600-fed OVX rats haveapproximately 50% less white adipose tissue mass compared toPhyto-Free-fed OVX rats, shown in FIG. 29, consistent with findings inExamples 9 and 10.

Serum leptin levels in Phyto-600-fed OVX rats are decreased byapproximately 30% compared to Phyto-Free-fed rats, shown in FIG. 30,reflecting the decreased white adipose tissue mass.

Example 11

Male and female Long-Evans rats are purchased from a supplier at 50 daysof age. All animals are raised (from conception to 50 days of age) onthe Phyto-200 diet. At 50 days old the male and female rats are randomlyassigned to one of four diet treatment groups: 1) AIN-76 diet containingapproximately <5 ppm isoflavones, 2) the Phyto-Free, 3) Phyto-200, or 4)Phyto-600 diet, described in previous examples. The AIN-76 diet containsextremely low concentrations of isoflavones, its formulation is quitedifferent compared to the other three diets. For example, the sucrosecontent is very high (almost approaching 50% of the total dietformulation) and has a dense white consistency that the rats may notenjoy consuming as much as the regular plant-based ingredient diets(i.e., the Phyto-Free diet uses corn and wheat in its formulation whichcontains very low levels of isoflavones); the Phyto-200 or Phyto-600diets use varying amount of soy meal in their formulations. The malerats are maintained on their assigned diets until 350 days of age(equivalent to middle-age in humans). The female rats are maintained onthe diets until 279 days of age (approaching middle-age in humans). Foodand water intake is measured to determine the potential influence ofthese parameters on body weight changes. In each case these factors donot contribute to the reductions in body weight with consumption of theisoflavone-containing diets (i.e., Phyto-200 and Phyto-600 diets; datanot shown).

Males—

At 112 days of age (on the diets for approximately 62 days), bodyweights are recorded, shown in FIG. 31. The males fed the Phyto-Freediet have the heaviest body weights and the Phyto-600-fed males have thelowest, while the males on the AIN-76 and the Phyto-200 diets fall inbetween these two group values. The Phyto-600-fed body weights aresignificantly lower, by approximately 14%, than the Phyto-Free-fedmales.

Correspondingly, at 279 and 350 days of age the male rats have a similarprofile to that observed at 112 days of age, shown in FIGS. 32 and 33,respectively. The males fed the Phyto-Free diet display the heaviestbody weights and the Phyto-600-fed males display the lowest bodyweights, while the males on the AIN-76 and Phyto-200 diets fall inbetween these two group values.

Males fed the AIN-76 or the Phyto-Phyto-Free fed males display thehighest white adipose tissue weights, measured at 350 days of age. ThePhyto-200-fed males show a 19% non-significant reduction in whiteadipose tissue weight compared to AIN-76 or Phyto-Free-fed rats. Malerats fed the Phyto-600 diet have significantly less adipose tissue mass,an approximate 40% reduction, compared to AIN-76 or Phyto-Free-fed rats,shown in FIG. 34.

Both serum insulin and leptin levels are significantly reduced as afunction of increasing concentrations of isoflavones in the diettreatments, shown in FIGS. 35 and 36, respectively. For example, malesfed the Phyto-200 or Phyto-600 diets have significant reductions ininsulin levels compared to AIN-76 fed males. Also, Phyto-600-fed malesshow an approximate 50% reduction in insulin levels compared toPhyto-Free-fed male Serum leptin profiles display a similar pattern tothat of the insulin results, where Phyto-200- or Phyto-600-fed maleshave significant reductions in serum insulin levels compared to eitherAIN-76 or Phyto-Free-fed males. Insulin levels in the Phyto-600-fedmales are 46% lower compared to the Phyto-200-fed males. However, thedifference between these two diet groups do not reach significance(p<0.065).

Females—

To determine the influence of the four diet treatments on body weight infemale rats, the body weights are measured at 112 and 279 days of age.At 112 days of age, females fed the Phyto-Free and the Phyto-200 dietshave the heaviest body weights and the Phyto-600-fed females have thelowest, while the AIN-76 diet group fall in between the values of theother three groups, shown in FIG. 37. Body weights of the Phyto-600-fedgroups are significantly lower, by approximately 10%, compared to thePhyto-Free- and the Phyto-200-fed females.

At 279 days of age, female rats have a similar profile to that ofage-matched males for changes in body weight as influenced by the diettreatments, shown in FIG. 38. Females fed the Phyto-600 diet display thelowest body weights compared to AIN-76 or Phyto-Free-fed groups. Thissignificant reduction in body weight in Phyto-600-fed females isapproximately 15% between the diet treatment groups tested.

Example 12

Noble rats were used to determine whether an inbred strain of rat hasbody and adipose tissue changes similar to those of out-bred strains ofrats such as the Long-Evans animals when placed on isoflavone-richdiets. Due to inbreeding, Noble rats are more fragile animals. Forexample, pregnant dams do not always carry their litters to term andfrequently have smaller litters. Noble rats have been used for more thantwenty years because they spontaneously generate tumors with aging,especially in hormonal-dependent organs of the reproductive tract. Thus,Noble rats have been extensively studied in the area of cancer research(e.g., R. L. Noble, Prostate carcinoma of the Nb rat in relation tohormones, Int Rev Exp Pathol, 1982, 23:113-159).

Male and female Noble rats are fed either the Phyto-Free or Phyto-600diets from conception until 145 days of age. Male Noble rats fed thePhyto-600 diet have significantly lower body weights at 145 days of agecompared to age-matched males fed the Phyto-Free diet, shown in FIG. 39.As previously observed for Long-Evans rats, the significant reduction inbody weight represents a modest but consistent decrease of approximately8% compared to Phyto-Free-fed males. In addition, white adipose tissuemass is significantly decreased in Phyto-600-fed males compared toPhyto-Free-fed, shown in FIG. 40.

Female Noble rats fed the Phyto-600 diet have a 6% reduction in bodyweight compared to Phyto-Free-fed females, shown in FIG. 41. Whiteadipose tissue mass is markedly decreased female Noble rats fed thePhyto-600 diet, shown in FIG. 42. The Phyto-600 diet group has a 61%reduction in adipose tissue compared to Phyto-Free-fed rats. Thedecrease in white adipose tissue is similar to that seen in Long-Evansrats.

Example 13

Prior to initiation of a Phyto-Free diet period Male Long-Evans rats arefed a Phyto-200 diet, as described in previous examples. The rats areplaced on a diet containing the Phyto-Free diet at approximately 52 daysof age and randomly assigned to three groups. Baseline body weightsafter 14 days and 21 days on the Phyto-Free diet for all rats aresimilar, shown in FIGS. 43 and 44, respectively. Beginning at 73 days ofage, rats receive daily subcutaneous 0.1 cc injections of vehicle(peanut oil), 1 milligram of a racemic mixture of equol in vehicle (0.83mg/kg body weight/day), or 5 milligrams of a racemic mixture of equol invehicle (4.2 mg/kg body weight/day) once every three days.

At 80 and 88 days of age, there are slight decreases in body weights andaverage body weight gains in both equol-injected groups compared tocontrols, however, these values are not significantly different fromcontrols, shown in FIGS. 45 and 46, respectively.

By the time the animals are 95 and 101 days of age, body weights areonly slightly decreased, ranging from 5 to 9% in equol-treated groups,shown in FIGS. 47 and 48. However, the average body weight gains inequol-injected animals at both 95 and 101 days are significantly reducedcompared to control values. Though body weight differences are notsignificant, adipose tissue deposition is strikingly lower inequol-treated groups. Adipose tissue mass in 101-day-old rats injectedwith equol is reduced by approximately 33% compared to controls, shownin FIG. 49.

To determine whether equol injections have an adverse effect on malereproductive organs, testis weights are quantified in these animals.There are no significant alterations in testes weight with the equolinjections, with testicular weight essentially the same among theinjection treatment groups, shown in FIG. 50.

Example 14

Fifty day-old Long-Evans males and females are caged individually andmaintained on a 10-hour dark 14-hour light schedule (lights on1400-0400). Animals are randomly assigned to diet groups, and allowed adlibitum access to one of four diet treatments: 1) AIN-76, 2) Phyto-Free,3) Phyto-200, or 4) Phyto-600 diet. The rats remain on the diets untilmid-aged (at approximately 300 days of age in males and at approximately330 days of age in females) when the animals are tested in the elevatedplus maze and anxiety-related behaviors were quantified. Thereafter,serum phytoestrogen levels are quantified by GC/MS according to themethod described by Coward L et al, J Agric Food Chem, 41:1961-1967. Thebehavioral patterns of anxiety are compared to the serum profiles ofcirculating isoflavone levels in the diet treatment groups by sex.

In males, there is a dose-dependent expression of anxiety-relatedbehaviors where animals fed the highest concentration of isoflavonesdisplay the lowest anxiety parameters. In contrast, animals fed theAIN-76 diet display the highest levels of anxiety, shown in FIG. 51.When the percent of time spent in the open arms is analyzed a similarpattern is seen to that of the number of entries into the open arms.Notably, the Phyto-600 fed males display the highest percentage of timespent in the open arms, while the lowest percentage of time spent in theopen arms is display by animals fed the AIN-76 diet, with Phyto-free andPhyto-200 values falling in between these maximal responses in adose-dependent fashion, shown in FIG. 52.

Prior to testing in the elevated plus maze, females are monitored byvaginal smears for 12 consecutive days to verify that none are cyclingto minimize effects of the estrous cycle. Female rats have a similarpattern of anxiety-related behaviors as those observed in the male rats.However, the influence of dietary isoflavones is not as robust as thatseen in males. Although, the highest percentage of the number of entriesinto (FIG. 53) or time spent on the open arms (FIG. 54) is seen inPhyto-600-fed females, with a stair-step pattern of decline until thelowest percentage of entries is seen in the AIN-76-fed females.

When behavioral testing is complete, the serum phytoestrogen levels aredetermined and compared to the patterns of anxiety-related behaviors. Inboth males (FIG. 55) and females (FIG. 56), the circulating isoflavonelevels correspond to the expression of anxiety-related behaviors,demonstrating an association between circulating isoflavone moleculesand anxiety. These data demonstrate that the isoflavone content of adiet can have significant effects on anxiety.

Example 15

Adult male Sprague-Dawley rats receive daily injections of either DMSO,racemic equol (0.250 mg/Kg/day), R-equol (0.250 mg/Kg/day), or S-equol(0.250 mg/Kg/day) in a total volume of 0.3 cc DMSO by subcutaneousinjection. At the end of seven consecutive days of treatment the animalsare tested in the elevated plus maze in order to quantifyanxiety-related behaviors. As shown in Table 5 below, males injectedwith racemic equol or R-equol display a significant decrease in anxietylevels compared to control rats.

TABLE 5 Anxiety-Related Behaviors in the Elevated Plus Maze of EquolInjected Male Rats. Injection Center Area Open Arm Time Open Arm EntriesTreatment Groups (in seconds) (in seconds) (in seconds) DMSO 17.5 + 4.116.3 + 4.0 1.0 + 0.03 Equol (racemic)  37.1 + 6.3*  40.9 + 7.0* 2.3 +0.4* R-Equol 36.4 + 7.6  50.0 + 10.0* 2.0 + 0.3* S-Equol 27.5 + 4.133.6 + 7.0  2.0 + 0.4** *= significant decrease in anxiety-relatedparameters (i.e., center area of maze time or time spent in the openarms or number of open arm entries) vs. control values. **= significantdecrease in anxiety-related behavior (i.e., number of open arm entries)vs. control values. n = 8 animals per group.

These findings are consistent with those obtained utilizing the 4dietary treatments containing different concentrations of isoflavones,and demonstrate that equol is a major factor in regulating anxiety andother neurological states such as mood and depression that have obviouspotential for broad health benefits.

Example 16

Twenty-nine (29) adults with hypercholesterolemia are fed a dietcontaining 33 mg of total isoflavones daily for 5 weeks. FIG. 57 showsthe observed change in BMI for each of the 29 individuals after 5 weeksof strict adherence to the diet containing isoflavones. The averagereduction in BMI over this period, although small, is neverthelesssignificant (p=0.01). These results suggest that phytoestrogen-richdiets can influence weight control in humans. The study did not identifythe component(s) responsible or the mechanism of weight control. Averagebaseline BMI (n=29) is 26.6±0.8, and the average BMI (n=29) at 5 weeksis 26.2±0.7.

While various embodiments of the present invention have been describedin detail, it will be apparent that further modifications andadaptations of the invention will occur to those skilled in the art. Itis to be expressly understood that such modifications and adaptationsare within the spirit and scope of the present invention.

1.-22. (canceled)
 23. A composition comprising: equol comprising aracemic mixture of R-equol and S-equol enantiomers; and at least one ofa pharmaceutically, nutraceutically, or cosmetically acceptable carrier,adjuvant, or excipient, wherein the composition is free from propyleneglycol.
 24. The composition of claim 23, wherein the carrier, adjuvantor excipient is nutraceutically acceptable.
 25. The composition of claim23, wherein the carrier, adjuvant or excipient is pharmaceuticallyacceptable.
 26. The composition of claim 23, wherein the carrier,adjuvant or excipient is cosmetically acceptable.
 27. A dietarysupplement comprising the composition of claim
 23. 28. A method fortreating or preventing a physiological or pathophysiological conditionsof benign prostatic hyperplasia and prostate cancer and mediated byandrogens in a mammal comprising: administering to the mammal aneffective amount of a composition comprising: equol comprising a racemicmixture of R-equol and S-equol enantiomers; and at least one of apharmaceutically, nutraceutically, or cosmetically acceptable carrier,adjuvant, or excipient, wherein the composition is free from propyleneglycol, thereby treating or preventing the physiological andpathophysiological conditions of benign prostatic hyperplasia andprostate.
 29. The method of claim 28, wherein the equol is in apharmaceutical.
 30. The method of claim 28, wherein the equol is in anover-the-counter medicament.
 31. The method of claim 28, wherein theequol is in a food product.
 32. The method of claim 28, wherein thecomposition comprises at least 1 mg equol for oral administration.
 33. Acomposition comprising: equol comprising a non-racemic mixture of equol,wherein the equol consists of more than 50 percent S-equol enantiomer;and at least one of a pharmaceutically, nutraceutically, or cosmeticallyacceptable carrier, adjuvant, or excipient, wherein the composition isfree from propylene glycol.
 34. The composition of claim 33, wherein theequol is in a pharmaceutical.
 35. The composition of claim 33, whereinthe equol is in an over-the-counter medicament.
 36. The composition ofclaim 33, wherein the equol is in a food product.
 37. The composition ofclaim 33, wherein the composition comprises at least 1 mg equol for oraladministration.
 38. The composition of claim 33, wherein the equol is ina dietary supplement.
 39. The composition of claim 33, wherein the equolis in a form of a topical application.
 40. The composition of claim 33,wherein the equol consists of 52 percent S-equol and 48 percent R-equol.